Using T cell receptor (TCR)-transgenic (N15 tg) Beta2-microglobulin deficient RAG2 minus/minus H-2b mice specific for the VSV8 (RGYVYQGL) octapeptide bound to Kb, we observed that a single weak agonist peptide variant of VSV8 (V4L), termed L4, induces T cell maturation while the cognate VSV8 peptide, in contrast, triggers negative selection. To characterize the molecular basis of this observation, a crystal structure of the L4/Kb complex was refined to 2.1 Angstrom units for comparison with the VSV8/Kb complex at a similar resolution. Aside from the methylene extension at the p4 position of L4 and resulting alteration of the exposed Kb Lys66 side chain, these structures are essentially identical yet result in very different selection outcomes. To now characterize the natural peptides involved in positive selection in vivo, four aims will be pursued. First, mass spectrometry approaches will be used to identify thymic Kb binding peptides which select the N15 receptor in N15tgRAG2 minus/minus Beta2M minus/minus, N15tgRAG2 minus/minus TAP1 minus/minus, TAP1 minus/minus and Beta2M minus/minus mice. Subsequently, deletion and/or mutation of the genes encoding the predominant naturally selecting peptides or exposure of thymic cultures to mAbs specific for a given peptide/MHC (pMHC) complex will allow us to ascertain whether there is an obligate requirement for a single peptide in the positive selection process. The effects of these natural peptides on selection of other VSV8/Kb-specific TCRs including N26 will be determined. Second, we will produce and utilize peptide/Kb tetramers to directly compare TCR affinities for cognate pMHC complexes and positively selecting pMHC complexes in pre-selected and post-selected thymocyte populations. The molecular features of the TCRs which interact with VSV8/Kb will be determined using single cell PCR-based methodologies. Assuming several natural peptides are capable of positively selecting VSV8/Kb specific T cells, comparision of the individual repertoires elicited will be made. Third, x-ray crystallographic techniques will be used to determine the structural basis of focal differences in the various positively selecting native pMHC complexes. Common features will be identified including any peptide-induced alterations of exposed MHC alpha-helical side chains. Fourth, the potential role for germline Valpha genes in positive selection will be assessed, with particular emphasis on CDR1 as suggested by our recent x-ray analysis of the N15 TCR-VSV8/Kb co- crystal.